Fig. 4 A (top row): T1 weighted MR images from planning software showing atlas-based localization of PPN: axial, coronal and sagittal. The axial section of the rostral PPN was taken at the level of the maximal inferior collicular diameter. Note that the contrast between white and gray matter seen on proton density images cannot be appreciated on T1 weighted MR images. B (bottom row): Axial MRI images through the inferior colliculi reformatted in a plane perpendicular to the midline of the fourth ventricular floor (VFL), (parallel to the BF line). The T1 image on the left shows excellent contrast between brain and CSF but little internal tissue contrast. The middle image is a Proton Density MRI sequence from the same patient and at an identical level. Contrast between gray and white matter structures is now readily apparent: gray matter appears hyperintense (bright); white matter appears hypointense (dark). Identifiable anatomical structures are labelled on the right. Note how the region of the PPN is of intermediate density between that of gray matter (e.g.: PAG) and white matter (e.g.: DSCP) perhaps in view of its reticular nature. CTT, central tegmental tract; DSCP, decussation of the superior cerebellar peduncles; LL, lateral lemniscus; ML, medial lemniscus; PAG, periaqueductal gray; PPN, pedunculopontine nucleus; PT, pyramidal tract; SN, substantia nigra; STT, spinothalamic tract. From: Stereotactic localization of the human pedunculopontine nucleus: atlas-based coordinates and validation of a magnetic resonance imaging protocol for direct localization Brain. 2008;131(6):1588-1598. doi:10.1093/brain/awn075 Brain | © The Author (2008). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Fig. 3 Atlas-based landmarks of the fourth ventricle as defined in Afshar's stereotactic atlas of the human brainstem and cerebellum. Variability in the angle of the mesencephalic flexure (a) may lead to increased variability of the spatial relationship of brainstem structures to the traditional AC-PC line. Brainstem structures may therefore enjoy a more constant relationship with fourth ventricular landmarks. A line is drawn tangential to the floor of the fourth ventricle in the midline (VFL); a second line passes through the fastigium, perpendicular to the first. The intersection of these two lines (B) and the fastigial point (F) define two points in a new reference plane in a similar manner to that defined by the more traditional AC-PC points. Extensions of the VFL and the AC-PC line subtend an angle a: the mesencephalic angle. From: Stereotactic localization of the human pedunculopontine nucleus: atlas-based coordinates and validation of a magnetic resonance imaging protocol for direct localization Brain. 2008;131(6):1588-1598. doi:10.1093/brain/awn075 Brain | © The Author (2008). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Fig. 2 Coronal sections taken 15. 5 mm (left) and 16 Fig. 2 Coronal sections taken 15.5 mm (left) and 16.5 mm (right) posterior to the midcommissural point (3.5 mm and 4.5 mm posterior to the posterior commissure). The PPN is labelled Tg.pdpo (nucleus tegmenti pedunculopontinus) and highlighted by the white arrow. In keeping with the reticular nature of the PPN, no clear boundaries are visible for this structure on this histological atlas. There is a 10 mm grid overlying the illustrations; the double horizontal line towards the top of the figures indicates the level of the mid anterior-posterior commissural point, that to the left of each figure indicates the midline. Selected abbreviations: A.aq, Annulus aquaeductus (periaqueductal grey); B.cj, Brachium conjunctivum (superior cerebellar peduncle); G.m, Corpus geniculatum mediale (medial geniculate body); L.l, Lemniscus lateralis (lateral lemniscus); L.m, Lemniscus medialis (medial lemniscus). Selected material taken and reprinted from Plate 29 of Schaltenbrand and Wahren (1977) with kind permission from Thieme. From: Stereotactic localization of the human pedunculopontine nucleus: atlas-based coordinates and validation of a magnetic resonance imaging protocol for direct localization Brain. 2008;131(6):1588-1598. doi:10.1093/brain/awn075 Brain | © The Author (2008). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Fig. 1 Axial sections perpendicular to long axis of brainstem Fig. 1 Axial sections perpendicular to long axis of brainstem. Row I: level of rostral PPN and mid inferior colliculi (IA and IB: 36 mm rostral to obex; IC and ID: 16 mm rostral to BF plane). Row II: through middle of PPN (IIA and IIB: 33 mm rostral to obex; IIC and IID: 13 mm rostral to BF plane). Row III: sections taken through caudal PPN (IIIA and IIIB: 31 mm rostral to obex; IIIC and IIID: 11 mm rostral to BF plane). Columns A and B: adapted from Paxinos et al. Column A presents negative photographs of sections stained with cresyl violet and acetylcholinesterase: white matter appears dark, gray matter appears light. Line drawings presented in column B show the outline of fibre tracts and nuclei. The ventricular system is solid black, a number of structures relevant to the PPN have been assigned a colour. Columns C and D: taken from Afshar et al. Column C presents photomicrographs of modified Mulligan stain sections; white matter tracts appear light, gray matter is dark. Column D: stereotactic drawings based upon probability data acquired from multiple hemi brainstems (see text and note superimposed stereotactic grid). Major structures are labelled but the PPN is not amongst them. Surrounding structures have been assigned the same colour scheme as in column B. Columns E and F: MRI images from a representative patient in the study acquired using the specifically modified proton density sequence described in the text. White matter appears hypointense and gray matter relatively hyperintense. Planning software was used to reconstruct axial images at a plane perpendicular to the midline of the fourth ventricular floor. Column F: interpretation of the structural arrangement of brainstem structures within the MR image assisted by the inherent image contrast as well as an understanding of the regional anatomy. The PPN can be directly localized within the gray matter lying lateral to the SCP and its decussation (green) and CTT (blue) and medial to the lemniscal systems (yellow) and is represented by a red circle. Note: atlas photographs and drawings reproduced here were originally published in conventional anatomical representation (anterior surface of the brainstem towards the bottom of the page). We have purposefully rotated these images through 180° to make their orientation analogous to the axial radiological imaging convention that is more familiar to clinicians. CTT/ctg, central tegmental tract; LL/ll, lateral lemniscus; ML, medial lemniscus; PPN, pedunculopontine nucleus; PPTgC, PPN pars compacta; PPTgD, PPN pars diffusa; SCP/scp, superior cerebellar peduncles; STT/spth, spinothalamic tract. Illustrations in columns A and B reprinted from Paxinos G, Huang XF: Atlas of the Human Brainstem. San Diego: Academic Press; 1995 with kind permission from Elsevier. Illustrations in columns C and D reprinted from Afshar et al. (1978) with kind permission from Lippincott Williams & Wilkins. From: Stereotactic localization of the human pedunculopontine nucleus: atlas-based coordinates and validation of a magnetic resonance imaging protocol for direct localization Brain. 2008;131(6):1588-1598. doi:10.1093/brain/awn075 Brain | © The Author (2008). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org